Primer and locked nucleic acid probe set for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of two types of goose parvovirus virulent and attenuated strains

Abstract

The invention relates to a primer and a locked nucleic acid probe set for real-time fluorescent quantitative PCR detection of two goose parvovirus virulent and attenuated strains, the goose parvovirus virulent and attenuated strains comprise MDGPV virulent and attenuated viruses and SBDSV virulent and attenuated viruses, and the sequences of the primer and the locked nucleic acid probe set are respectively shown as SEQ ID NO.1-8. The LNA-TaqMan real-time fluorescent quantitative PCR detection method established by utilizing the primers and the LNA-TaqMan probe group can be used for simultaneously detecting strong and weak goose parvovirus strains (strong and weak muscovy duck gosling plague virus and / or strong and weak duck short beak and dwarf syndrome virus), identifying, diagnosing and accurately quantifying, and is high in sensitivity, strong in specificity and good in repeatability.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to primers and locked nucleic acid (LNA)-TaqMan probe sets for fluorescent quantitative PCR detection of strong and weak strains of two goose parvoviruses, namely Muscovy duck-origingoose parvovirus (MDGPV) and short beak and dwarfism syndrome-goose parvovirus (SBDSV). Background Art

[0002] Goose parvovirus (GPV) infection can cause an acute, contagious, septicemic disease that primarily affects goslings and Muscovy ducklings aged 4 to 20 days. It can easily lead to acute enteritis and inflammation of organs such as the heart, liver, and kidneys. The disease is highly contagious, with high morbidity and mortality rates, and occurs sporadically, causing significant economic losses to the poultry industry and severely restricting its healthy development. GPV, a member of the genus Dependoviridae in the family Parvoviridae, is a nonenveloped, single-stranded DNA virus with roughly equal numbers of positive-strand and negative-strand DNA particles. Its genome is approximately 5.0 kb in size and consists of two open reading frames (ORFs). The left ORF (LORF) encodes the nonstructural proteins (NS1 and NS2), while the right ORF (RORF) encodes the three structural proteins (viral capsid proteins (VP)) VP1, VP2, and VP3. The NS and VP proteins share a common carboxyl terminus, forming a nested structure. Like other autonomous parvoviruses, the GPV NS protein is involved in regulating viral replication and its pathogenesis, while the VP protein is responsible for packaging single-stranded viral progeny DNA to form infectious progeny virions. Since 1997, outbreaks of Muscovy duck-origin goose parvovirus (MDGPV) have occurred in Muscovy duck breeding areas such as Putian and Fuqing in Fujian Province. The disease is characterized by varying degrees of diarrhea, fibrinous necrotic enteritis, and mucosal sloughing and embolism in the duck intestine. MDGPV is a naturally recombinant strain of classical goose parvovirus (C-GPV) and Muscovy duck parvovirus (MDPV), which causes "three-week disease" in Muscovy ducks. It can be transmitted through direct or indirect contact and vertical transmission. In 2015, short beak and dwarfism syndrome (SBDSV), a distantly related strain of MDGPV, was reported in my country. SBDSV has a wider range of natural hosts and can cause disease in commercial meat ducks, including Cherry Valley ducks, Peking ducks, Muscovy ducks, and Taiwan white ducks. Onset ranges from 6 to 40 days of age, with a 5% to 20% incidence rate and a low mortality rate. Most affected ducks die of stunted ducklings. Clinically, Muscovy duck gosling plague and short beak and dwarfism syndrome (SBDSV) often occur in farmed waterfowl in the same areas, often exhibiting mixed infections and facilitating frequent recombination and mutation of waterfowl parvovirus. Compared to virulent strains, these variants exhibit reduced virulence, resulting in atypical clinical symptoms in Muscovy ducks, making differential diagnosis possible only through molecular biological methods.

[0003] There are many methods for GPV detection, including virus isolation and identification, ELISA, indirect immunofluorescence assay (IFA), serum neutralization test (SN), real-time fluorescence quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and other methods. However, in actual production applications, most methods can only detect but cannot distinguish between strong and weak GPV strains. The use of molecular biology techniques such as fluorescence quantitative PCR and gene sequencing is currently the most effective method for distinguishing strong and weak virus strains. Recent studies have found that modifying conventional TaqMan probes can effectively improve the specificity and sensitivity of real-time fluorescence quantitative PCR detection methods.

[0004] Locked nucleic acid (LNA) is a chemical modification that occurs through a 2-methyl sugar linkage between O2 and C4. LNA modification enhances the stability and affinity of DNA molecules in PCR reactions. It has been reported that the melting temperature of the oligonucleotide increases by 96°C with each additional nucleotide insertion. This mutational modification enables significant amplification of the assay environment and identification of single-base mismatches. Furthermore, it allows for more complex experiments to be performed in a single test tube. The unique design of LNA makes it stable in DNA or RNA environments, particularly with its strong DNA / RNA recognition and binding abilities. Therefore, selectively modifying some bases in conventional TaqMan probes with LNA improves sensitivity (specificity) for single-base mismatches, facilitates design, and enhances signal-to-noise ratios, in part due to reduced fluorescence from spurious binding and the close proximity of the quencher and reporter dye. This technology has been widely used in pathogen detection, pathogen identification, and gene mutation detection. Currently, there are no reports in China on LNA-TaqMan probes for waterfowl parvovirus fluorescence quantitative PCR.

[0005] Fluorescence quantitative PCR detection methods based on gene sequences encoding functional protein coding regions are currently the primary method for distinguishing different strains of waterfowl parvovirus. In light of this, the present invention establishes fluorescence quantitative detection methods for the VP1 gene using LNA-TaqMan qPCR, specifically targeting MDGPV and SBDSV strains, capable of distinguishing between strong and weak MDGPV and strong and weak SBDSV strains, respectively. This approach aims to provide a new method option for the early diagnosis and quantitative detection of MDGPV and SBDSV. Summary of the Invention

[0006] To achieve the above-mentioned purpose, the present invention has developed LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe groups for two strong and weak strains of goose parvovirus, wherein the strong and weak strains of goose parvovirus include strong and weak strains of MDGPV and strong and weak strains of SBDSV; the primers and LNA-TaqMan probe group are suitable for fluorescence quantitative PCR differential diagnosis of strong and weak strains of MDGPV and strong and weak strains of SBDSV in samples.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] To achieve the above objectives, the present invention provides primers and locked nucleic acid probe sets for LNA-TaqMan real-time fluorescence quantitative PCR detection of two strong and weak strains of goose parvovirus, the two goose parvoviruses including Muscovy duck gosling plague virus (MDGPV) and duck short beak dwarf syndrome virus (SBDSV);

[0009] The primer and locked nucleic acid probe set includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the virulent Muscovy duck gosling plague virus and the attenuated Muscovy duck gosling plague virus, respectively. The specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0010] MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3' (SEQ ID NO. 1), MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3' (SEQ ID NO. 2);

[0011] PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB (SEQ ID NO.3),

[0012] D-Probe: VIC-ACCCCGACgAAAA-MGB (SEQ ID NO.4);

[0013] The primer and locked nucleic acid probe set also includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak syndrome virus, respectively. The specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0014] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3' (SEQ ID NO.5),

[0015] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3' (SEQ ID NO. 6);

[0016] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB (SEQ ID NO.7),

[0017] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB (SEQ ID NO. 8).

[0018] The present invention also provides a kit for detecting strong and weak strains of goose parvovirus by LNA-TaqMan real-time fluorescence quantitative PCR, wherein the strong and weak strains of goose parvovirus include strong and weak strains of Muscovy duck gosling plague virus, and the kit includes reaction system A;

[0019] The reaction system A includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the virulent Muscovy duck gosling plague virus and the attenuated Muscovy duck gosling plague virus, respectively. The specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0020] MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3',

[0021] MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3';

[0022] PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB,

[0023] D-Probe: VIC-ACCCCGACgAAAA-MGB.

[0024] The reaction system A is 20 μL, and each 20 μL of reaction system A includes:

[0025] IIProbe qPCR SuperMix 10μL, primers MDXPV-121F and MDXPV-121R at a concentration of 0.4μmol / L each, probe PT-Probe 0.4μL at a concentration of 0.2μmol / L, probe D-Probe 1.0μL at a concentration of 0.5μmol / L, template DNA 2.0μL, Passive Reference Dye II (50×) 0.5μL, and nuclease-free water to 20μL.

[0026] The goose parvovirus strong and weak strains also include duck short beak dwarf syndrome virus strong and weak strains, and the kit also includes a reaction system B;

[0027] The reaction system B includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak and dwarf syndrome virus, respectively. The specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0028] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3',

[0029] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3';

[0030] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB,

[0031] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

[0032] The reaction system B is 20 μL, and each 20 μL of reaction system B includes:

[0033] IIProbe qPCR SuperMix 10μL, primers SBDSV-124F and SBDSV-150R (both at a concentration of 0.4μmol / L), 0.8μL of the M15-Probe probe (at a concentration of 0.4μmol / L), 0.6μL of the M15F92-Probe probe (at a concentration of 0.3μmol / L), 2.0μL of template DNA, 0.5μL of Passive Reference Dye II (50×), and nuclease-free water to 20μL. The reaction conditions for the LNA-TaqMan real-time fluorescence quantitative PCR method in this kit are: 94°C for 30s, followed by 40 cycles of 94°C for 5s and 60°C for 30s with fluorescence collection.

[0034] The present invention also provides a kit for detecting strong and weak strains of goose parvovirus by LNA-TaqMan real-time fluorescence quantitative PCR, wherein the strong and weak strains of goose parvovirus include strong and weak strains of duck short-beak syndrome virus; the kit includes reaction system B;

[0035] The reaction system B includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak and dwarf syndrome virus, respectively. The specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0036] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3',

[0037] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3';

[0038] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB,

[0039] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

[0040] The reaction system B is 20 μL, and each 20 μL of reaction system B includes:

[0041] IIProbe qPCR SuperMix 10 μL, primer SBDSV-124F and primer SBDSV-150R (both at a concentration of 0.4 μmol / L) 0.8 μL each, probe M15-Probe (at a concentration of 0.4 μmol / L) 0.8 μL, probe M15F92-Probe (at a concentration of 0.3 μmol / L) 0.6 μL, template DNA 2.0 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free water to 20 μL;

[0042] The reaction conditions of the LNA-TaqMan real-time fluorescence quantitative PCR method of the kit are: 94° C. for 30 s; 94° C. for 5 s, 60° C. for 30 s to collect fluorescence, and 40 cycles.

[0043] Compared with the prior art, the advantages of the present invention are:

[0044] 1. Simultaneous detection, rapid detection, and high efficiency: The LNA-TaqMan real-time fluorescence quantitative PCR detection method established by the present invention using the primers and LNA-TaqMan probe set can simultaneously detect, differentially diagnose, and accurately quantify strong and weak strains of goose parvovirus (strong and weak strains of Muscovy duck gosling plague virus and / or duck short-beak syndrome virus), simplifying the operating procedures and saving costs. At the same time, this detection method does not require conventional agarose gel electrophoresis detection. After the reaction is completed, the results can be determined using the program provided by the real-time fluorescence quantitative PCR machine.

[0045] 2. Accurate quantification: By preparing standard samples and drawing standard curves, the strong and weak toxins of Muscovy duck gosling plague virus and duck short beak and stunted syndrome virus in the samples to be tested can be directly and accurately quantified based on the Ct values of the strong and weak toxins of the Muscovy duck gosling plague virus and duck short beak and stunted syndrome virus in the samples to be tested.

[0046] 3. High sensitivity: The method established in the present invention can detect the template of the MDGPV virulent VP1 gene recombinant standard plasmid pUC57-PTVP1 with a minimum detection amount of 2.0×10 0 The minimum detection amount of the template of the MDGPV attenuated VP1 gene recombinant standard plasmid pUC57-DVP1 was 2.0×10 1 copies / μL, indicating that the dual fluorescence quantitative LNA-TaqMan PCR method established by the present invention based on primers MDXPV-121F, primers MDXPV-121R, probes PT-Probe, and probes D-Probe has high sensitivity; the minimum detection amount of the template of the SBDSV virulent VP1 gene recombinant standard plasmid pUC57-M15VP1 established by the present invention is 1.0×10 0 The minimum detection amount of the template of the SBDSV attenuated VP1 gene recombinant standard plasmid pUC57-M15F92VP1 was 1.0×10 1 The results showed that the dual fluorescence quantitative LNA-TaqMan PCR method established by the present invention using primers SBDSV-124F, primers SBDSV-150R, probes M15-Probe and probes M15F92-Probe had high sensitivity.

[0047] 4. High Specificity: A dual real-time fluorescence quantitative LNA-TaqMan PCR method, established and optimized using reaction system A, was used to detect nucleic acids from pUC57-PTVP1 and pUC57-DVP1 positive plasmids, as well as MDGPV PT, MDGPV D, MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. The results showed amplification curves for nucleic acids from the virulent MDGPV PT and the weak MDGPV D, as well as a mixture of their plasmid standards. No amplification curves were observed for nucleic acids from other pathogens or the negative control. This demonstrates the high specificity of this method and its ability to differentiate between strong and weak MDGPV strains.

[0048] A dual LNA-TaqMan real-time fluorescence quantitative PCR method, established and optimized using reaction system B, was used to detect nucleic acids from pUC57-M15VP1 and pUC57-M15F92VP1 plasmids, as well as nucleic acids from SBDSV-M15, SBDS-M15F92, MDPV, C-GPV, MDGPV, DEV, DPMV, DHV-I, and DTMUV. The results showed amplification curves for nucleic acids from both virulent SBDSV-M15 and weaker SBDS-M15F92, as well as a mixture of their plasmid standards. No amplification curves were observed for nucleic acids from other pathogens or the negative control. This demonstrates the high specificity of this method and its ability to differentiate between strong and weak SBDSV strains.

[0049] 5. Good repeatability: The fluorescent quantitative LNA-TaqMan PCR method established in the present invention has good repeatability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the standard curve of LNA-TaqMan dual real-time fluorescence quantitative PCR in Example 1, wherein A is the standard curve of MDGPV-PTVP1 gene dual real-time fluorescence quantitative LNA-TaqMan PCR, and B is the standard curve of MDGPV-D VP1 gene dual real-time fluorescence quantitative LNA-TaqMan PCR.

[0051] Figure 2 It is the specificity of the LNA-TaqMan dual real-time fluorescence quantitative PCR in Example 1; wherein, 1: pUC57-PTVP1 plasmid standard; 2: MDGPV PT strong strain; 3: pUC57-DVP1 plasmid standard; 4: MDGPV D weak strain; 5-11: MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0052] Figure 3 Figure 1 is the sensitivity result of LNA-TaqMan dual real-time fluorescence quantitative PCR in Example 1, where A is the sensitivity test result of dual real-time fluorescence quantitative LNA-TaqMan PCR of VP1 gene of MDGPV PT strain (1 to 7: the concentration of pUC57-PTVP1 recombinant plasmid is 2.0×10 6 copies / μL~2.0×10 0 copies / μL; 8: negative control); B is the sensitivity test result of dual real-time fluorescence quantitative LNA-TaqMan PCR of VP1 gene of MDGPV D strain (1-7: the concentration of pUC57-DVP1 recombinant plasmid was 2.0×10 6 copies / μL~2.0×10 0copies / μL; 8: negative control).

[0053] Figure 4 It is the standard curve of the dual real-time fluorescence quantitative LNA-TaqMan PCR in Example 2, wherein A is the standard curve of the dual real-time fluorescence quantitative LNA-TaqMan PCR of SBDSV-M15 VP1 gene, and B is the standard curve of the dual real-time fluorescence quantitative LNA-TaqMan PCR of SBDSV-M15F92 VP1 gene.

[0054] Figure 5 This is a graph showing the specific results of the LNA-TaqMan dual real-time fluorescence quantitative PCR in Example 2; wherein, 1: pUC57-M15VP1 plasmid standard; 2: SBDSV-M15 virulent strain; 3: pUC57-M15F92VP1 plasmid standard; 4: SBDSV-M15F92 weak strain; 5-11: MDPV, MDGPV, C-GPV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0055] Figure 6 is the sensitivity result of LNA-TaqMan dual real-time fluorescence quantitative PCR in Example 2; wherein A is the sensitivity test result of LNA-TaqMan dual real-time fluorescence quantitative PCR of VP1 gene of SBDSV-M15 strain (1 to 7: the concentration of pUC57-M15VP1 recombinant plasmid is 1.0×10 6 copies / μL~1.0×10 0 copies / μL; 8: negative control); B is the sensitivity test result of LNA-TaqMan double real-time fluorescence quantitative PCR of VP1 gene of SBDSV-M15F92 strain (1-7: the concentration of pUC57-M15F92VP1 recombinant plasmid was 1.0×10 6 copies / μL~1.0×10 0 copies / μL; 8: negative control). DETAILED DESCRIPTION

[0056] To further clarify the objectives, technical solutions, and advantages of the present invention, embodiments of the present invention are described in further detail below with reference to the accompanying drawings. Materials and instruments not described herein are conventional in the art, and operational details not described herein are conventional in the art. The nucleic acid sequences shown herein are written from left to right in a 5' to 3' orientation.

[0057] The upstream in the present invention refers to the 5' end or the 5' end direction of the nucleic acid, and the downstream refers to the 3' end or the 3' end direction of the nucleic acid.

[0058] Example 1: Establishment of a fluorescent quantitative LNA-TaqMan PCR differential diagnosis method for strong and weak Muscovy duck and gosling plague viruses 1 Materials and methods

[0059] 1.1 Viruses and Pathogens

[0060] The virulent strain of MDGPV-PT, the weak strain of MDGPV-D, MDPV, C-GPV, SBDSV, duck plague virus, duck paramyxovirus, duck hepatitis virus type 1, and duck Tembusu virus are all stored in the laboratory of the Fujian Academy of Agricultural Sciences. The specimens were liver, spleen, pancreas, kidney, and other tissue samples collected from a Muscovy duck farm in Fujian Province suspected of being infected with gosling plague since 2023 and stored in the laboratory of the Fujian Academy of Agricultural Sciences.

[0061] 1.2 Main reagents and instruments

[0062] IIProbe qPCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd. FastPure Viral DNA / RNA Mini Kit (RC311-01), FastPure Gel DNA Extraction Mini Kit, and FastPure Plasmid Mini Kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd. A fluorescence quantitative PCR instrument (ABI7500) was purchased from Roche.

[0063] 1.3 Primer design and synthesis

[0064] The VP1 sequences of the genomes of eight MDGPV lineage virus strains included in GenBank were analyzed using DNAStar software. A pair of universal primers for MDGPV and two specific LNA-TaqMan probes for strong and weak MDGPV were designed using the online primer design tool Primer-BLAST. The probes were labeled with different luminescent groups to enable them to distinguish between strong and weak strains. The sequences of primers and LNA-TaqMan probes are shown in Table 1 (Note: lowercase letters represent LNA modified bases).

[0065] Table 1 Fluorescence quantitative LNA-TaqMan PCR primer and probe sequences

[0066]

[0067] 1.4 Viral nucleic acid extraction

[0068] Nucleic acid was extracted from MDGPV-PT, MDGPV-D, C-GPV, MDPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV according to the instructions of the FastPure Viral DNA / RNA Extraction Kit. RNA was reverse transcribed into cDNA and stored at -20°C along with viral DNA until use.

[0069] 1.5 Construction of recombinant plasmid standards

[0070] The variable region gene fragments of the VP1 gene of the virulent PT and attenuated D strains of MDGPV were synthesized and cloned into the pUC57 vector to construct the recombinant plasmid standards pUC57-PTVP1 and pUC57-DVP1. Gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The recombinant plasmid concentration was determined using a full-wavelength microplate reader, and the copy number (copies / μL) was calculated. The results showed that the pUC57-PTVP1 and pUC57-DVP1 were 6.0×10 9 copies / μL, 8.0×10 9 Copies / μL. Both were diluted to 2.0×10 9 copies / μL and stored at -20°C as recombinant plasmid standards for future use.

[0071] 1.6 Optimization of LNA-TaqMan Real-time Fluorescence Quantitative PCR Method

[0072] Equal volumes of two recombinant plasmid standards, pUC57-PTVP1 and pUC57-DVP1, were mixed and used as templates. Fluorescence quantitative PCR amplification was performed in the same system (i.e., reaction system A) using two specific primers (MDGPV-121F and MDGPV-121R) and LNA-TaqMan probes (PT-Probe and D-Probe). The TaqMan real-time fluorescence quantitative PCR reaction system was set to 20 μL. The matrix method was used to optimize the annealing temperature (56°C, 57°C, 58°C, 59°C, 60°C), final primer concentrations (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1.0 μmol / L), and final probe concentrations (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L) to obtain the optimal reaction conditions for TaqMan real-time fluorescence quantitative PCR.

[0073] 1.7 Establishment of the standard curve for LNA-TaqMan real-time fluorescence quantitative PCR

[0074] The pUC57-PTVP1 and pUC57-DVP1 plasmid standards were diluted 10-fold to obtain a final concentration of 2.0×10 6 copies / μL-2.0×10 0 A mixture of plasmid standards with a concentration of 100 copies / μL was used as a template, Nuclease-free Water was set as a negative control, and optimized TaqMan real-time fluorescence quantitative PCR was used for amplification. A standard curve was drawn with the logarithmic value of the plasmid copy number at different concentrations as the X-axis and the corresponding Ct value as the Y-axis.

[0075] 1.8 Specificity test of LNA-TaqMan real-time fluorescence quantitative PCR method

[0076] The optimized MDGPV strong and weak virulence dual TaqMan real-time fluorescence quantitative PCR method was used to detect the MDGPV strong and weak virulence using DNA of MDGPV-PT, MDGPV-D, MDPV, C-GPV, SBDSV, and DEV and cDNA of DPMV, DHV-I, and DTMUV stored in our laboratory, a mixture of pUC57-PTVP1 and pUC57-DVP1 plasmid standards as a positive control, and Nuclease-free water as a negative control. 1.9 Sensitivity test of the LNA-TaqMan real-time fluorescence quantitative PCR method

[0077] The two recombinant plasmid standards pUC57-PTVP1 and pUC57-DVP1 were diluted 10-fold and mixed in equal volumes to obtain a final concentration of 2.0×10 6 copies / μL-2.0×10 0 A mixture of plasmid standards with a concentration of 100 copies / μL was used as a template for amplification using the optimized dual TaqMan fluorescent quantitative PCR to evaluate the sensitivity of the method.

[0078] 1.10 Repeatability test of LNA-TaqMan real-time fluorescence quantitative PCR method

[0079] Three concentrations of pUC57-PTVP1 (2.0×10 6 copies / μL, 2.0×10 5 copies / μL, 2.0×10 4 copies / μL), pUC57-DVP1 (2.0×10 6 copies / μL, 2.0×10 5 copies / μL, 2.0×10 4Copies / μL) were used as templates, and the optimized dual TaqMan fluorescence quantitative PCR method was used to perform intra-batch and inter-batch reproducibility tests. In the intra-batch reproducibility test, three replicates were set for each sample, and in the inter-batch reproducibility test, samples were tested at three different time periods. The results were statistically analyzed to verify the reproducibility of the method.

[0080] 1.11 Testing of clinical samples

[0081] Twenty-five tissue samples, including liver, spleen, pancreas, and kidney, collected from a Muscovy duck farm in Fujian Province suspected of being infected with gosling plague since 2023, were ground and subjected to viral nucleic acid extraction using an appropriate amount of supernatant as directed by the nucleic acid extraction kit. MDGPV was detected using an optimized duplex LNA-TaqMan real-time fluorescence quantitative PCR method. Amplified positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the agreement between the PCR method and sequencing results was calculated.

[0082] 2 Results

[0083] 2.1 Optimization of reaction conditions for LNA-TaqMan real-time fluorescence quantitative PCR

[0084] After optimization experiments, the optimal reaction system for dual LNA-TaqMan real-time fluorescence quantitative PCR targeting MDGPV-PT and MDGPV-D VP1 genes (reaction system A) was determined: IIProbe qPCR SuperMix (10 μL), upstream-specific PCR primer MDDPV-121F (0.4 μmol / L), downstream-specific PCR primer MDDPV-121R (0.4 μmol / L) (0.8 μL each), probe PT-Probe (0.2 μmol / L) (0.4 μL), probe D-Probe (0.5 μmol / L) (1.0 μL), recombinant plasmid standard mixture template DNA (2.0 μL), Passive Reference Dye II (50×) (0.5 μL), and nuclease-free water to 20 μL. The optimal reaction conditions for the LNA-TaqMan real-time fluorescence quantitative PCR assay targeting the MDGPV PT and MDGPV D VP1 genes were: 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s with fluorescence collection.

[0085] 2.2 Establishment of standard curve

[0086] The recombinant plasmid standards of pUC57-PTVP1 and pUC57-DVP1 were diluted 10-fold (2.0×10 6 copies / μL~2.0×10 0Copies / μL) were mixed as templates and amplified using the optimized dual LNA-TaqMan real-time fluorescence quantitative PCR method. The logarithm of the number of plasmid starting templates was used as the X-axis and the cycle threshold (Ct value) was used as the Y-axis to draw a standard curve. The results showed that ( Figure 1 ), the recombinant plasmid standard pUC57-PTVP1 was at 2.0×10 6 copies / μL~2.0×10 0 copies / μL, pUC57-DVP1 at 2.0×10 6 copies / μL~2.0×10 0 The copies / μL showed a good linear relationship at their respective Ct values. The standard curve of pUC57-PTVP1 was Y = -3.4X + 17.045, and the correlation coefficient was R 2 =0.995, the amplification efficiency was 96.858; the standard curve of pUC57-DVP1 was Y=-3.303X+17.545, and the correlation coefficient R 2 =0.994, and the amplification efficiency is 100.779.

[0087] 2.3 Specificity test results

[0088] An optimized dual real-time fluorescence quantitative LNA-TaqMan PCR method was used to detect nucleic acids from pUC57-PTVP1 and pUC57-DVP1 positive plasmids, as well as MDGPV PT, MDGPV D, MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. The results showed that nucleic acids from the virulent MDGPVPT and the weakly virulent MDGPV D, as well as a mixture of their plasmid standards, showed amplification curves, while nucleic acids from other pathogens and negative controls showed no amplification curves. Figure 2 ). This shows that this method has high specificity and can achieve differential diagnosis of strong and weak MDGPV strains.

[0089] 2.4 Sensitivity test results

[0090] The established dual real-time fluorescence quantitative LNA-TaqMan PCR method for the strong and weak VP1 genes of MDGPV was used to perform sensitivity detection on a mixed sample of the recombinant standard plasmids pUC57-PTVP1 and pUC57-DVP1. The results showed that the minimum detection amount of the template of the recombinant standard plasmid pUC57-PTVP1 of the strong and weak VP1 gene of MDGPV by the established method was 2.0×10 0 copies / μL( Figure 3 A) The minimum detection amount of the template for the MDGPV attenuated VP1 gene recombinant standard plasmid pUC57-DVP1 was 2.0×10 1 copies / μL( Figure 3B) This indicates that the dual fluorescence quantitative LNA-TaqMan PCR method established in this experiment has high sensitivity.

[0091] 2.6 Repeatability test results

[0092] Using optimized reaction conditions, three dilutions of pUC57-PTVP1 and pUC57-DVP1 plasmid standards were used as templates for testing. Three intra- and inter-assay replicates were performed for each template dilution. The results showed that both the intra- and inter-assay coefficients of variation for the same template at different concentrations were within 2.0% (Table 2), demonstrating the excellent reproducibility and stability of the developed fluorescent quantitative LNA-TaqMan PCR method.

[0093] Table 2 Repeatability results of LNA-TaqMan fluorescence quantitative PCR

[0094]

[0095] 2.7 Test results of clinical samples

[0096] This study collected 25 clinical samples suspected of being infected with Muscovy duck and gosling plague and tested them using the established duplex real-time fluorescence PCR technique. Two samples of virulent MDGPV were detected, with a positive rate of 8.0%. No virulent MDGPV was detected. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequence alignment using BLAST online software showed 100% concordance between the real-time fluorescence LNA-TaqMan PCR assay established in this study and the sequencing results. This demonstrates that the established MDGPV fluorescence quantitative LNA-TaqMan PCR assay has high sensitivity and robustness, making it suitable for testing clinical samples.

[0097] Example 2: Establishment of a fluorescent quantitative LNA-TaqMan PCR differential diagnosis method for strong and weak duck short-beak syndrome

[0098] 1 Materials and Methods

[0099] 1.1 Viruses and clinical samples

[0100] The virulent M15 strain of SBDSV, the attenuated M15F92 strain of SBDSV, Muscovy duck parvovirus, goose parvovirus, Muscovy duck gosling plague virus, duck plague virus, duck paramyxovirus, duck hepatitis virus type 1, and duck Tembusu virus are all stored in the laboratory of the Fujian Academy of Agricultural Sciences. The specimens were liver, spleen, pancreas, kidney, and other tissue samples collected from a duck farm suspected of having short-beaked duck syndrome in Fujian Province between 2023 and 2024 and stored in the laboratory of the Fujian Academy of Agricultural Sciences.

[0101] 1.2 Main reagents and instruments

[0102] IIProbe qPCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd. FastPure Viral DNA / RNA Mini Kit (RC311-01), FastPure Gel DNA Extraction Mini Kit, and FastPure Plasmid Mini Kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd. A fluorescence quantitative PCR instrument (ABI7500) was purchased from Roche.

[0103] 1.3 Primer design and synthesis

[0104] The VP1 sequences of the genomes of 15 SBDSV lineage virus strains included in GenBank were analyzed using DNAStar software. A pair of universal primers for SBDSV and two specific probes for strong and weak SBDSV virulence were designed using the online primer design tool Primer-BLAST. The probes were labeled with different luminescent groups to enable them to distinguish between strong and weak strains. The primer and probe sequences are shown in Table 3 (Note: lowercase letters represent LNA modified bases).

[0105] Table 3 Fluorescence quantitative LNA-TaqMan PCR primer and probe sequences

[0106]

[0107] 1.4 Viral nucleic acid extraction

[0108] Nucleic acid was extracted from SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, DEV, DPMV, DHV-I, and DTMUV according to the instructions of the FastPure Viral DNA / RNA Extraction Kit. RNA was reverse transcribed into cDNA and stored at -20°C along with viral DNA until use.

[0109] 1.5 Construction of recombinant plasmid standards

[0110] The variable region gene fragments of the VP1 gene of the virulent M15 and attenuated M15F92 strains of SBDSV were synthesized and cloned into the pUC57 vector to construct the recombinant plasmid standards pUC57-M15VP1 and pUC57-M15F92VP1. Gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The recombinant plasmid concentration was determined using a full-wavelength microplate reader, and the copy number (copies / μL) was calculated. The results showed that the pUC57-M15VP1 and pUC57-M15F92VP1 had a concentration of 2.25×10 9copies / μL, 1.75×10 9 Copies / μL. Both were diluted to 1.0×10 9 copies / μL and stored at -20°C as recombinant plasmid standards for future use.

[0111] 1.6 Optimization of LNA-TaqMan Real-time Fluorescence Quantitative PCR Method

[0112] Equal volumes of two recombinant plasmid standards, pUC57-M15VP1 and pUC57-M15F92VP1, were mixed and used as templates. Fluorescence quantitative PCR amplification was performed in the same system (i.e., reaction system B) using two specific primers (SBDSV-124F and SBDSV-150R) and LNA-TaqMan probes (M15-Probe and M15F92-Probe). The TaqMan real-time fluorescence quantitative PCR reaction system was set to 20 μL. The matrix method was used to optimize the annealing temperature (56°C, 57°C, 58°C, 59°C, 60°C), final primer concentrations (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1.0 μmol / L), and final probe concentrations (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L) to obtain the optimal reaction conditions for LNA-TaqMan real-time fluorescence quantitative PCR.

[0113] 1.7 Establishment of the standard curve for LNA-TaqMan real-time fluorescence quantitative PCR

[0114] pUC57-M15VP1, pUC57-M15F92VP1 and plasmid standard were diluted 10-fold to obtain a final concentration of 1.0×10 6 copies / μL-1.0×10 0 A plasmid standard mixture with 100 copies / μL was used as a template, and Nuclease-free water was set as a negative control. Optimized LNA-TaqMan real-time fluorescence quantitative PCR was used for amplification. A standard curve was drawn with the logarithmic value of the plasmid copy number at different concentrations as the X-axis and the corresponding Ct value as the Y-axis.

[0115] 1.8 Specificity test of LNA-TaqMan real-time fluorescence quantitative PCR method

[0116] The optimized dual real-time fluorescence quantitative LNA-TaqMan PCR method for MDGPV strong and weak virulence was used to detect the DNA of SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, DEV and cDNA of DPMV, DHV-I, and DTMUV stored in our laboratory, a mixture of pUC57-M15VP1 and pUC57-M15F92VP1 plasmid standards as a positive control, and Nuclease-free water as a negative control.

[0117] 1.9 Sensitivity test of LNA-TaqMan real-time fluorescence quantitative PCR method

[0118] The two recombinant plasmid standards pUC57-M15VP1 and pUC57-M15F92VP1 were diluted 10-fold and mixed in equal volumes to obtain a final concentration of 1.0×10 6 copies / μL-1.0×10 0 A mixture of plasmid standards with a concentration of 100 copies / μL was used as a template for amplification using the optimized dual fluorescent quantitative LNA-TaqMan PCR to evaluate the sensitivity of the method.

[0119] 1.10 Repeatability test of LNA-TaqMan real-time fluorescence quantitative PCR method

[0120] Three concentrations of pUC57-M15VP1 (1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL), pUC57-M15F92VP1 (1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 Copies / μL) were used as templates, and the optimized dual fluorescence quantitative LNA-TaqMan PCR method was used to perform intra-batch and inter-batch reproducibility tests. In the intra-batch reproducibility test, three replicates were set for each sample, and in the inter-batch reproducibility test, samples were tested at three different time periods. The results were statistically analyzed to verify the reproducibility of the method.

[0121] 1.11 Testing of clinical samples

[0122] Twenty clinical tissue samples from the duck farm were ground and processed. Viral nucleic acid was extracted using an appropriate amount of supernatant as directed by the nucleic acid extraction kit. SBDSV was detected using an optimized dual real-time fluorescence quantitative LNA-TaqMan PCR assay. Amplified positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the agreement between the PCR assay and sequencing results was calculated.

[0123] 2 Results

[0124] 2.1 Optimization of reaction conditions for real-time fluorescence quantitative LNA-TaqMan PCR

[0125] After optimization experiments, the optimal dual TaqMan real-time fluorescence quantitative PCR reaction system for SBDSV-M15 and SBDSV-M15F92 VP1 genes was determined (reaction system B): II Probe qPCR SuperMix (10 μL), upstream-specific PCR primer SBDSV-124F (0.4 μmol / L), downstream-specific PCR primer SBDSV-150R (0.4 μmol / L) (0.8 μL each), probe M15-Probe (0.4 μmol / L) (0.8 μL), probe M15F92-Probe (0.3 μmol / L) (0.6 μL), recombinant plasmid standard mixture template DNA (2.0 μL), Passive Reference Dye II (50×) (0.5 μL), and nuclease-free water to 20 μL. The optimal reaction conditions for TaqMan real-time fluorescence quantitative PCR for both the SBDSV-M15 and SBDSV-M15F92 VP1 genes were: 94°C for 30 s, followed by 94°C for 5 s and 60°C for 30 s with fluorescence collection, for 40 cycles.

[0126] 2.2 Establishment of standard curve

[0127] The recombinant plasmid standards of pUC57-M15VP1 and pUC57-M15F92VP1 were diluted 10-fold (1.0×10 6 copies / μL~1.0×10 0 The optimized dual real-time fluorescence quantitative LNA-TaqMan PCR method was used for amplification. The logarithm of the number of plasmid starting templates was used as the X-axis and the cycle threshold (Ct value) was used as the Y-axis to draw a standard curve. The results showed that ( Figure 4 ), the recombinant plasmid standard pUC57-M15VP1 was 1.0×10 6 copies / μL~1.0×10 0 copies / μL, pUC57-M15F92VP1 at 1.0×106 copies / μL~1.0×10 0 The copies / μL showed a good linear relationship at their respective Ct values. The standard curve of pUC57-M15VP1 was Y=-3.584X+16.46, and the correlation coefficient was R 2 =0.996, the amplification efficiency was 99.112; the standard curve of pUC57-M15F92VP1 was Y=-3.289X+13.974, and the correlation coefficient R 2 =0.998, and the amplification efficiency is 101.393.

[0128] 2.3 Specificity test results

[0129] The optimized dual LNA-TaqMan real-time fluorescence quantitative PCR method was used to detect the nucleic acids of pUC57-M15VP1, pUC57-M15F92VP1 positive plasmids and SBDSV-M15, SBDS-M15F92, MDPV, C-GPV, MDGPV, DEV, DPMV, DHV-I, and DTMUV. The results showed that ( Figure 5 ), nucleic acids from both the virulent SBDSV-M15 and the attenuated SBDSV-M15F92, as well as a mixture of their plasmid standards, showed amplification curves, while other pathogen nucleic acids and the negative control showed no amplification curves. This demonstrates that this method is highly specific and can differentiate between virulent and attenuated SBDSV strains.

[0130] 2.4 Sensitivity test results

[0131] The established dual real-time fluorescence quantitative LNA-TaqMan PCR method for the strong and weak VP1 genes of SBDSV was used to perform sensitivity detection on a mixed sample of the recombinant standard plasmids pUC57-M15VP1 and pUC57-M15F92VP1. The results showed that the minimum detection amount of the template of the recombinant standard plasmid pUC57-M15VP1 of the strong and weak VP1 gene of SBDSV by the established method was 1.0×10 0 copies / μL( Figure 6 A) The minimum detection amount of the template of the SBDSV attenuated VP1 gene recombinant standard plasmid pUC57-M15F92VP1 established by the method is

[0132] 1.0×10 1 copies / μL( Figure 6 B) This indicates that the dual fluorescence quantitative LNA-TaqMan PCR method established in this experiment has high sensitivity.

[0133] 2.6 Repeatability test results

[0134] Using optimized reaction conditions, three dilutions of pUC57-M15VP1 and pUC57-M15F92VP1 plasmid standards were used as templates for testing. Three intra- and inter-assay replicates were performed for each template dilution. The results showed that both the intra- and inter-assay coefficients of variation for the same template at different concentrations were within 2.0% (Table 4), demonstrating the excellent reproducibility and stability of the developed fluorescence quantitative PCR method.

[0135] Table 4 Repeatability results of LNA-TaqMan fluorescence quantitative PCR

[0136]

[0137] 2.7 Test results of clinical samples

[0138] This study collected 20 clinical samples suspected of short-beak duck syndrome and tested them using the established duplex real-time fluorescence PCR technique. Two samples of virulent SBDSV were detected, with a positive rate of 10%. No samples of weak SBDSV were detected. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequence alignment using BLAST online software showed 100% concordance between the real-time fluorescence LNA-TaqMan PCR assay established in this study and the sequencing results. This demonstrates that the SBDSV quantitative fluorescence LNA-TaqMan PCR assay established in this study has high sensitivity and strong stability, making it suitable for testing clinical samples.

Claims

1. A primer and locked nucleic acid probe set for LNA-TaqMan real-time fluorescence quantitative PCR detection of two strong and weak strains of goose parvovirus, characterized by: The two goose parvoviruses include Muscovy duck gosling plague virus and duck short-beak syndrome virus; The primers and locked nucleic acid probe sets include LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the virulent Muscovy duck gosling plague virus and the attenuated Muscovy duck gosling plague virus, respectively, and their specific sequences are as follows: MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3', MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3'; PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB, D-Probe: VIC-ACCCCGACgAAAA-MGB; The primer and locked nucleic acid probe set also includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak syndrome virus, respectively, and their specific sequences are as follows: SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3', SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3'; M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB, M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

2. A kit for detecting strong and weak strains of goose parvovirus by LNA-TaqMan real-time fluorescence quantitative PCR, characterized in that: The goose parvovirus strong and weak strains include Muscovy duck gosling plague virus strong and weak strains, and the kit includes reaction system A; The reaction system A includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the virulent Muscovy duck gosling plague virus and the attenuated Muscovy duck gosling plague virus, respectively, and their specific sequences are as follows: MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3', MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3'; PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB, D-Probe: VIC-ACCCCGACgAAAA-MGB.

3. The kit according to claim 2, wherein: The reaction system A is 20 μL, and each 20 μL of reaction system A includes: 2.0× IIProbe qPCR SuperMix 10μL, primers MDXPV-121F and MDXPV-121R at a concentration of 0.4μmol / L each, probe PT-Probe 0.4μL at a concentration of 0.2μmol / L, probe D-Probe 1.0μL at a concentration of 0.5μmol / L, template DNA 2.0μL, Passive Reference Dye II (50×) 0.5μL, and nuclease-free water to 20μL.

4. The kit according to claim 2, wherein: The goose parvovirus strong and weak strains also include duck short beak dwarf syndrome virus strong and weak strains, and the kit also includes a reaction system B; The reaction system B includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak and dwarf syndrome virus, respectively, and their specific sequences are as follows: SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3', SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3'; M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB, M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

5. The kit according to claim 4, wherein: The reaction system B is 20 μL, and each 20 μL of reaction system B includes: 2.0× IIProbe qPCR SuperMix 10 μL, primers SBDSV-124F and SBDSV-150R (both at a concentration of 0.4 μmol / L), 0.8 μL each, M15-Probe (at a concentration of 0.4 μmol / L), M15F92-Probe (at a concentration of 0.3 μmol / L), 0.6 μL, template DNA 2.0 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free water to 20 μL. The kit according to claim 2 is characterized in that the reaction conditions for the LNA-TaqMan real-time fluorescence quantitative PCR method in the kit are: 94°C for 30 s, followed by fluorescence collection at 94°C for 5 s and 60°C for 30 s, for 40 cycles.

6. A kit for detecting strong and weak strains of goose parvovirus by LNA-TaqMan real-time fluorescence quantitative PCR, characterized by: The goose parvovirus strong and weak strains include duck short-beak dwarf syndrome virus strong and weak strains; the kit includes reaction system B; The reaction system B includes LNA-TaqMan real-time fluorescence quantitative PCR detection primers and LNA-TaqMan probe sets for the strong and weak strains of duck short-beak and dwarf syndrome virus, respectively, and their specific sequences are as follows: SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3', SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3'; M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB, M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

7. The kit according to claim 6, characterized in that: The reaction system B is 20 μL, and each 20 μL of reaction system B includes: 2.0× IIProbe qPCR SuperMix 10 μL, primer SBDSV-124F and primer SBDSV-150R (both at a concentration of 0.4 μmol / L) 0.8 μL each, probe M15-Probe (at a concentration of 0.4 μmol / L) 0.8 μL, probe M15F92-Probe (at a concentration of 0.3 μmol / L) 0.6 μL, template DNA 2.0 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free water to 20 μL; The reaction conditions of the LNA-TaqMan real-time fluorescence quantitative PCR method of the kit are: 94° C. for 30 s; 94° C. for 5 s, 60° C. for 30 s to collect fluorescence, and 40 cycles.

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